The first step of sensory perception is the transduction of physical stimuli into cellular signaling events. The excitatory cation channel TRPV1 is expressed on peripheral nerve fibers and is activated by noxious heat and acidic pH, thereby serving as a physiological detector of harmful external conditions. The Julius lab recently discovered that at least one species of spider, Psalmopoeus cambridgei, produces peptide toxins that elicit pain and inflammation through activation of TRPV1. As highly selective modulators of particular channel-types, peptide toxins have proven to be powerful tools for understanding the structure, function, and physiology of several ion channel families. In this proposal both biochemical and electrophysiological techniques will be used to investigate the mechanism of toxin activation of TRPV1. These studies will advance our molecular understanding of this important sensory transducer and generate valuable biochemical probes for the channel. At the basic science level, this project will bring insight into the molecular underpinnings of this important ion channel and advance our molecular understanding of how noxious stimuli are detected in the peripheral nervous system. These efforts will contribute, in the long term, towards understanding and controlling acute and chronic pain syndromes. The project has two specific aims. The first aim is to determine the sites responsible for toxin activation of TRPV1. Chimeras and point mutations will be generated from TRPV1 and mutant channels will be tested for toxin activation. Channel activation will be assayed by both calcium imaging and electrophysiology. Also, fluorescent- or radio-labeled derivatives of the vanillotoxins will be generated to directly monitor toxin binding. The second aim is to characterize a novel TRPV1 toxin. I have discovered a novel toxin agonist of TRPV1 that exhibits strikingly little sequence homology with the previously identified toxins. I hypothesize that despite the differences in sequence, this novel toxin and the known toxins have evolved convergently to target the same region of TRPV1. Also, the new toxin has a unique sequence, unlike any sequence that has been reported for a peptide toxin, and how this unique sequence dictates toxin function will be investigated. This project will work toward a mechanistic understanding of how components in tarantula venom interact with the capsaicin receptor, TRPV1, in order to cause pain and inflammation. This work will advance our understanding of the molecular mechanisms that underly sensation of noxious stimuli. In the long-term, this study will contribute towards understanding and controlling acute and chronic pain syndromes. My long-term career goal is to make significant contributions to the scientific understanding of neural signaling at the molecular level. With this proposed research project, I will study ion channel physiology to gain an intimate understanding of the techniques and approaches used in molecular neuroscience, and I will also become familiar with the logic and approaches necessary for productive independent research. Specifically, I will use both biochemical and electrophysiological techniques to study the interactions of a channel-toxin complex. I have some training in the biochemistry of protein-protein interactions, and I will use the proposed project to apply this background to the membrane environment. I will also expand my technical skillset, with both membrane-specific and general biochemical skills. Another portion of the research plan involves electrophysiological techniques, which are exceptionally important for studying neural signaling. I will utilize a variety of recording configurations to integrate my scholastic understanding of electrophysiological theory. The expertise of members of the Julius lab and neighboring labs represents a great asset in developing these skills. This combination of biochemical and electrophysiological techniques and perspectives represents a solid foundation from which to pursue my goals of conducting independent research on signaling molecules in the nervous system.